Source: Bhushan, S. et al., Isolation of Sertoli Cells and Peritubular Cells from Rat Testes. J. Vis. Exp. (2016).
This video demonstrates the isolation procedure of Sertoli cells from mouse seminiferous tubules, a component of the testes. Enzymatic digestion and repeated washes promote the release of Sertoli cells into the solution, which can be cultured further for proliferation and growth.
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of Media, Enzyme Solutions, and Animals
2. Preparation of Seminiferous Tubules
3. Removal/Isolation of Peritubular Cells (PTCs)
4. Isolation of Sertoli Cells (SCs)
Figure 1. Testes from Wistar rats are excised and decapsulated. (A) The peritoneal cavity is opened through a longitudinal incision as described in the text. An asterisk (*) indicates the epidydimal fat pad. (B) Testes are removed by cutting the spermatic cord and (C) collected in a 50 ml conical tube containing 20 ml PBS. When all testes have been collected they are disinfected in 20 ml of 1% (w/v) iodine in ethanol. For removal of the iodine, they are quickly washed twice with 25 ml PBS each. (D) Testes after first PBS wash. (E) Testes are transferred to a Petri dish (on the right), and seminiferous tubules are squeezed out of the opened tunica albuginea by means of closed scissor blades (on the left) as described in the text. (F) The decapsulated testes still form a compact testicle-shaped mass with single tubules protruding.
Figure 2. Isolation of Sertoli cells. (A) After removal of interstitial cells by Trypsin-DNase I digestion and washing 9x with PBS tubules become mobilized and look clean. (B) During collagenase-hyaluronidase-DNase I treatment peritubular cells from the outer layer and germ cells are released. The tubules get shortened and obtain a rough appearance. (C) Tubular fragments after washing 4x with PBS. (D) Hyaluronidase-DNase I treatment releases residual peritubular cells as well as germ cells. Tubules get further shortened, and tubular aggregates form. (E) Tubular aggregates after washing 4x with PBS. (F) Single Sertoli cells are produced by passing the aggregates 10x through an 18G needle. Scale bars in (A-E) = 200 µm, in (F) = 50 µm.
Figure 3. Immunofluorescence staining. Staining of purified primary peritubular cells with actin (smooth muscle) antibody (A) and of Sertoli cells with vimentin antibody (B). No contaminating cells are visible in both fields of view. Scale bar in (A) and (B) = 10 µm. (C) and (D) Electron microscopic pictures of Sertoli cells after 4 days in culture. (C) Note the close contact of adjacent cells (arrowheads) that leads to the cobblestone-like pattern. A single lipid droplet (L) is observed in the cytoplasm of each cell. (D) Most abundant organelles are mitochondria (M) and the Golgi apparatus (G). The nucleus (N) contains a nucleolus (Nc) and shows the typical fissure-like indentation. Scale bar in (C) = 1 µm and in (D) = 0.25 µm.
The authors have nothing to disclose.
Corning™ Cell Strainers 70 µm | Corning | 431751 | White color |
DAPI mountant ProLong® Gold Antifade | Life Technologies | P-36931 | |
D-glucose | Sigma | G8644 | 100 g/l |
Dulbecco's PBS without Ca2+/Mg2+ | Gibco | 14190-094 | |
DNase I | Roche | 10104159001 | |
Hyaluronidase from bovine testis | Sigma | H3506 | |
Penicillin (5,000 U/ml) Streptomycin (5,000 µg/ml) | Gibco | 15070-063 | 100x solution |
RPMI-1640 | Gibco | 21875-034 | Contains 300 mg/L L-glutamine |
Trypsin from porcine pancreas | Sigma | T5266 | |
Trypan Blue Stain (0.4%) | Gibco | 15250-061 | |
Trypsin inhibitor from soybean | Sigma | T6522 | The Sigma product is considerably cheaper than the previously used BPTI (Aprotinin) from Roche. |
Wistar WU rats | Charles River | N/A | Should be 19 days old on day of experiment. |